JP2008044912A - Antitumor agent, hyaluronidase inhibitor or therapeutic agent of atopic dermatitis originated from laminaria angustata var. longissima - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antitumor agent, a hyaluronidase inhibitor or a therapeutic agent of atopic dermatitis, containing fucoidan obtained from an extract of seaweeds as an active ingredient, and utilizing the bioactivities thereof. <P>SOLUTION: The antitumor agent, the hyaluronidase inhibitor or the therapeutic agent of the atopic dermatitis contains the fucoidan obtained from Laminaria angustata var. longissima as an active ingredient. The fucoidan is obtained by extracting the Laminaria angustata var. longissima with a diluted aqueous hydrochloric acid solution, and concretely, is one or all of four fractions obtained by extracting the Laminaria angustata var. longissima with the diluted aqueous hydrochloric acid solution, converting the extract into a cetylpyridinium chloride complex of a polysaccharide, and subjecting the cetylpyridinium chloride complex of the polysaccharide to a DEAE-sepharose column. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an antitumor agent, a hyaluronidase inhibitor or a therapeutic agent for atopic dermatitis comprising fucoidan extracted from Nagacomb as an active ingredient.

  The Okinawa region has historically created a unique culture of food, clothing and shelter in Japan, and this uniqueness is considered to be a major factor in the longevity of people living in the Okinawa region. Among these, there are many unique Okinawan food cultures, especially because they use a lot of seaweeds such as kombu and mozuku.

In recent years, it has been found that fucoidan and other sulfated polysaccharides contained in these seaweeds have various physiological activities, attracting attention as useful biological resources, and various studies have been conducted. This fucoidan is one of the sulfated polysaccharides abundantly contained in mozuku, kombu, wakame, hijiki, etc., and in addition to L-fucose, D-galactose, D-xylose and D-glucuronic acid are used as constituent sugars. Furthermore, it is a polysaccharide having a sulfate group bonded with an ester.
Up to now, fucoidan has been found to have an anti-cholesterol action, an anticoagulant action, etc., and has already been developed and marketed as a health supplement material.

  The present inventors have focused on polysaccharides contained in seaweeds, and identified and identified functional polysaccharides from polysaccharides contained in seaweeds that naturally grow in Okinawa, and their gelation mechanism and tumor cells. Has been reported on the apoptosis-inducing effect (see Non-Patent Document 1). In addition, chemical properties of alginic acid from Nagacomb have been reported (see Non-Patent Document 2).

  Furthermore, the present inventors have a molecular weight of 500,000 from Okinawa mozuku (Cladosiphon okamuranus TOKIDA), and the composition ratios of L-fucose, D-xylose, D-glucuronic acid, acetic acid and sulfuric acid are 3.0 to 4. We have found acetylfucoidan which is 0, 0.1-0.3, 0.8-1.2, 0.5-1.0, 0.8-1.2 and have already obtained a patent (Patent Literature) 1), this acetylfucoidan has been found to have excellent antitumor activity, and a patent application has been filed (see Patent Document 2).

In order to investigate the possibility of using seaweed for various pharmaceuticals, the present inventors have extensively studied the physiological activity of such seaweed extracts, particularly fucoidan.
Japanese Patent No. 3371124 Japanese Patent Application No. 2005-236456 Masakuni Tada, et al., "Applied Glycoscience" 43, 143-148 (1996) Tayuki, Nakayama, "Abstracts of Annual Meeting of the Japanese Society of Applied Glycoscience", p. 30, Kagoshima, (2004)

  That is, an object of the present invention is to provide various therapeutic agents using fucoidan from an extract of a certain kind of seaweed as an active ingredient and utilizing its physiological activity.

  The present inventors have conducted extensive studies on the physiological activity of various seaweed extracts, and found that fucoidan obtained by extraction from Nagacomb exhibits excellent physiological activity and completed the present invention.

  That is, the present invention provides an antitumor agent, a hyaluronidase inhibitor, or a therapeutic agent for atopic dermatitis characterized by comprising fucoidan obtained from Nagacomb as an active ingredient.

  The present invention also provides an antitumor agent, a hyaluronidase inhibitor, or an atopic dermatitis therapeutic agent, wherein fucoidan is obtained by extracting Nagacomb with dilute hydrochloric acid aqueous solution.

  Further, the present invention relates to four fractions obtained by fucoidan extracting Nagacomb with dilute hydrochloric acid solution to obtain cetylpyridinium chloride complex polysaccharide, and subjecting the cetylpyridinium chloride complex polysaccharide to a DEAE-Sepharose column. An antitumor agent, a hyaluronidase inhibitor, or an atopic dermatitis therapeutic agent characterized by comprising any or all of them.

  Furthermore, this invention provides the food-drinks characterized by containing the said anti-tumor agent, a hyaluronidase inhibitor, or an atopic dermatitis therapeutic agent.

  An antitumor agent comprising fucoidan obtained from Nagacomb of the present invention as an active ingredient has excellent antitumor activity, and a hyaluronidase inhibitor comprising fucoidan obtained from Nagacomb as an active ingredient also exhibits excellent hyaluronidase inhibitory activity. Furthermore, a therapeutic agent for atopic dermatitis comprising fucoidan obtained from Nagacomb as an active ingredient relieves the symptoms of atopic dermatitis and exhibits an excellent therapeutic effect on atopic dermatitis.

  Therefore, an antitumor agent, a hyaluronidase inhibitor or an atopic dermatitis therapeutic agent containing fucoidan obtained from Nagacomb of the present invention as an active ingredient exhibits excellent antitumor activity and hyaluronidase inhibitory activity, respectively. It can be used for treatment of tumors, treatment of atopic dermatitis, etc., and by taking this as a food and drink containing these therapeutic agents on a daily basis, it can prevent tumors and generate atopic dermatitis. It can also be suppressed.

  The antitumor agent, hyaluronidase inhibitor or atopic dermatitis therapeutic agent of the present invention comprises fucoidan which is an acidic polysaccharide obtained from Nagacomb as an active ingredient, and more specifically, Nagacomb is extracted with dilute hydrochloric acid aqueous solution. Fucoidan obtained as an active ingredient.

  Nagambu (Laminaria angustata var. Longissima), which is a raw material, is a kind of seaweed belonging to the family of brown algae. As seaweeds belonging to the same Compositae family, there are macomb, resilive, hosome-comb, etc., in addition to Nagacomb, but those that can be used for the purpose of the present invention are Nagacomb. The growing zones are the east of Kushiro Port, Akkeshi, Hamaka, Hanasaki, Hokkaido, and the Pacific coast, Tomojima Islands, Kunigo Island, and Etorofu Island. Although it is considered to be a variety of honey beetle, it has very long leaves compared to honey beetle, and the leaf length is 7 to 8 m on average and 15 to 20 m at maximum. The morphological features are a long, strip-shaped leaf, a gently wavy edge, a narrow middle strip, and a rounded wedge base.

The antitumor agent, hyaluronidase inhibitor, or therapeutic agent for atopic dermatitis of the present invention comprises fucoidan, which is a kind of acidic polysaccharide obtained by extracting Nagacomb with an acidic aqueous solution such as dilute hydrochloric acid.
More specifically, fucoidan obtained from this Nagacomb was extracted with an acidic aqueous solution such as dilute hydrochloric acid, then precipitated as a cetylpyridinium chloride complex polysaccharide, dissolved in an aqueous calcium chloride solution, and treated with decetylpyridinium. Thereafter, any or all of the four fractions obtained by applying to a DEAE-Sepharose column are included. Fucoidan obtained from Nagacomb is a novel fucoidan containing L-fucose as a main component and replacing a large amount of sulfuric acid, and is generally composed of L-fucose, D-galactose, D-xylose, D-glucuronic acid and sulfuric acid. In terms of L-fucose at a ratio (mass ratio) of 4.0, the composition is as shown in Table 1 below.

  Specifically, a method for obtaining fucoidan used in the present invention from Nagacomb is as follows. First, the dried alga body of Nagacomb is pulverized and then added to an aqueous hydrochloric acid solution and stirred for several hours to overnight. Next, this is filtered, the filtrate is passed through a column such as Celite 545 layer, and the resulting extract is precipitated with cetylpyridinium chloride (hereinafter referred to as “CPC”) to obtain a CPC complex precipitate. The precipitate of the CPC complex, which is a partially purified acidic polysaccharide, was dissolved in a calcium chloride solution and applied to a DEAE-Sepharose column, whereby four fractions of fucoidan having the composition shown in Table 1 above (fractions 1 to fraction). Minute 4) can be obtained.

  The antitumor agent of the present invention can be produced by formulating fucoidan obtained from Nagacomb as it is or in combination with a known appropriate pharmaceutical carrier. The content of fucoidan obtained from Nagacomb in the antitumor agent of the present invention is not particularly limited as long as it exhibits an antitumor activity. For example, fucoidan obtained from Nagacomb is 20 to 80% by mass, preferably 50 to 50% by mass. 80% by mass.

  The antitumor agent of the present invention is effective against tumors such as leukemia, lymphoma, gastric cancer, liver cancer, lung cancer, breast cancer and the like, and administration methods according to these tumors, for example, oral administration, local administration, etc. Can be selected. In addition, the dosage form of these antitumor agents is not particularly limited, and depending on the administration method, oral agents such as tablets, granules, granules, powders, capsules, and parenterals such as injections and infusions. It can be used as an agent. Furthermore, the dose and the number of administrations may be determined according to a normal antitumor agent.

  Furthermore, in the antitumor agent of the present invention, the antitumor activity can be further enhanced by using another antitumor agent in combination. Other such anti-tumor agents that can be used in combination include cytarabine, melphalan, cyclophosphamide, mechloretamine, carmustine, methotrexate, mercaptopurine, fluorouracil, dactinomycin, bleomycin, daunorubicin, adriamycin, idarubicin , Mitomycin, cisplatin, vinblastine, vincristine, paclitaxel, docetaxel, siglitazone, pioglitazone, troglitazone, rosiglitazone, fluorouracil, acegraton, aclarubicin, fadrazole, asparaginase, busulfan, carmofur, carboplatin, carboplatin, carboplatin, carboplatin , Epirubicin, epithiostanol, et Poside, flutamide, goserelin, hydroxycarbamide, medroxyprogesterone, irinotecan, ifosfamide, krestin, leuprorelin, melphalan, mepithiostan, mestanolone, mitoxantrone, mitotane, neocalcinostatin, nedaplatin, nimustine, nitrogen mustard, Examples include oxymetholone, picibanil, pentostatin, pepleomycin, pirarubicin, procarbazine, ranimustine, zobuzoxan, tamoxifen, thiotepa, tegafur, tretinoin, toremifene, bestatin, tegafur uracil, vindesine, dinostatin, dexamethasone, prednisolone, etc. Of these, particularly excellent effects can be obtained when combined with adriamycin, mitomycin, etoposide, vindesine, cyclophosphamide, cytarabine and the like.

  The hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention can also be produced by formulating fucoidan obtained from Nagacomb as it is or in combination with a known appropriate pharmaceutical carrier. The content of fucoidan obtained from Nagacomb in the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention is not particularly limited as long as it is an amount exhibiting hyaluronidase inhibitory activity and allergy suppressing effect. For example, fucoidan obtained from Nagacomb Is 20 to 80% by mass, preferably 50 to 80% by mass.

  As described above, fucoidan, which is a sulfated polysaccharide obtained from Nagacomb, has antitumor activity and hyaluronidase inhibitory activity, and such sulfated polysaccharide has an allergy suppressing effect and antitumor effect as a food factor. Therefore, the antitumor agent, hyaluronidase inhibitor or atopic dermatitis therapeutic agent of the present invention can be contained in ordinary food and drink and taken daily. Such a method for producing a food or drink may be obtained by adding an effective amount of the antitumor agent, hyaluronidase inhibitor or atopic dermatitis therapeutic agent of the present invention to the raw material of the food or drink, etc. What is necessary is just to follow the manufacturing method of goods.

  Examples of these foods and drinks include beverages such as tea beverages and nutritional supplements, and foods such as dietary supplements such as capsules, tablets, and powders.

  EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples. Further, “%” and “part” in the examples are based on mass unless otherwise noted.

(1) Extraction of partially purified sulfated polysaccharide from Nagacomb The dried alga of Hokkaido Nagacomb is stirred overnight at room temperature in a 0.1M aqueous hydrochloric acid solution to 10%, centrifuged, and the supernatant is removed. Suction filtered. The filtrate was neutralized with 0.1M sodium hydroxide and then concentrated. The precipitate was washed with ethanol, dehydrated and dried under reduced pressure to obtain a crude polysaccharide. The obtained crude polysaccharide was dissolved in distilled water, insoluble matters were centrifuged, and then passed through Celite 545 layer. An equal amount of 2% cetylpyridinium chloride (CPC) was added to the filtrate and left overnight at room temperature to form a white precipitate of CPC complex polysaccharide. After centrifuging the white precipitate of the crude polysaccharide, the precipitate was washed with distilled water, and 10 times its volume of 4M calcium chloride solution was added and completely dissolved at 40 ° C. Four times the amount of ethanol was added to this solution, and the resulting precipitate was centrifuged, washed several times with ethanol, and dissolved in distilled water. After sufficient dialysis, freeze-dried partially purified sulfated polysaccharide was obtained.

(2) Production of purified sulfated polysaccharide (purified fucoidan) A DEAE-Sepharose FF column (Cl ⁻ ) obtained by equilibrating the partially purified sulfated polysaccharide obtained in (1) above with 20 mM Tris-HCl buffer (pH 8.0). Mold, manufactured by Amersham Bioscience). Elution was performed with a linear gradient (final concentration 1.7 M NaCl) between 20 mM Tris-HCl buffer (pH 8.0) and 20 mM Tris-HCl buffer (pH 8.0) containing 1.9 M NaCl. The column flow rate was 0.5 mL / min, fractionated in 5 mL increments, and sugar was quantified by the phenol sulfate method and the carbazole sulfate method. The eluate was concentrated and freeze-dried. The lyophilized product was dissolved in distilled water, desalted through ion-exchange resin (Bio-Gel P4), and the fraction detected near the void volume of the column was dialyzed and freeze-dried. A purified sulfated polysaccharide (purified fucoidan) consisting of four fractions was obtained. Each fraction was dialyzed, freeze-dried, desalted with a Bio-GelP4 column, and freeze-dried to obtain a purified polysaccharide fraction. These were designated as fraction 1, fraction 2, fraction 3, and fraction 4, respectively. The yields of these purified polysaccharide fractions were 0.09%, 0.18%, 0.11% and 0.10% for fraction 1, fraction 2, fraction 3, and fraction 4, respectively (dry Alga body ratio).

  Each fraction of these purified polysaccharides was subjected to a liquid chromatograph, and the chemical composition was determined from the liquid chromatogram. As a result, the fraction 1 coincided with the retention times of L-fucose, D-galactose and D-xylose, and from the area ratio, the constituent sugar ratio was L-fucose: D-galactose: D-xylose = 67. 2: 15.9: 16.9. Similarly, fraction 2 coincided with the retention time of L-fucose and D-galactose, and the constituent sugar ratio was L-fucose: D-galactose = 96.0: 4.0. Fraction 3 coincided with the retention time of L-fucose and D-galactose, and the constituent sugar ratio was L-fucose: D-galactose = 47.6: 52.4. Similarly, in the fraction 4, the constituent sugar ratio was L-fucose: D-galactose = 26.6: 73.4.

Further, when the amount of glucuronic acid was measured using D-glucuronic acid as a sample, fractions 1, 2, 3, and 4 were 21.5%, 2.6%, and 4.3%, respectively. And 4.8%. When the sulfuric acid content was determined by the barium sulfate gelatin method, it was 27.5%, 45.6%, and 31.5 for fraction 1, fraction 2, fraction 3, and fraction 4, respectively, in terms of SO ₃ Na. 1% and 38.4%.

  From the above, the total sugar amount excluding glucuronic acid was assigned to the constituent sugar ratio of each neutral sugar, and the sugar constituent ratio (%) and sulfuric acid content ratio (%) of the purified polysaccharide were determined. The results are shown in Table 2.

(3) Evaluation of Hyaluronidase Inhibitory Activity Hyaluronidase inhibitory activity was measured for fraction 1, fraction 2, fraction 3 and fraction 4 of the purified polysaccharide obtained from Nagacomb obtained in (2) above.
Each fraction of purified polysaccharide serially diluted from 0 to 50 μg / mL and hyaluronidase were preincubated for 20 minutes at room temperature. Hyaluronidase used was isolated from the testes of the cow (Sigma). Next, NaCl as a hyaluronidase activator and hyaluronic acid derived from human heso (MP Biomedicals) as a hyaluronidase activator were added to this solution, reacted at 37 ° C. for 40 minutes, heated to 100 ° C., and then Mogan-Elson method. Was quantified.

The inhibitory activity of each fraction at each dilution concentration is shown in FIG. Fraction 1, fraction 2, fraction 3 and fraction 4 all showed hyaluronidase inhibitory activity in a concentration-dependent manner. For comparison, a heparin (Celsus Laboratory), for which hyaluronidase inhibitory activity has already been found, was used as a positive control to carry out an inhibition test in the same manner. The results are also shown in FIG.
From this result, fraction 1 was inhibited 70% at 25-50 μg / mL, and the other fractions were inhibited approximately 90% at 25 μg / mL.

Next, a 50% inhibitory concentration (IC ₅₀ ) was determined from the results of FIG. 1, and the results are shown in FIG. The IC ₅₀ of fraction 1, fraction 2, fraction 3 and fraction 4 were 22 μg / mL, 12 μg / mL, 15.2 μg / mL and 15.2 μg / mL, respectively. The IC ₅₀ of heparin used as a control was 12.5 μg / mL. Fraction 2 showed activity inhibition comparable to that of heparin, and other fractions showed activity inhibition equivalent to that of heparin.

Evaluation of anti-tumor activity of fucoidan obtained from Nagacomb (1) Measurement of U937 cell growth inhibitory effect Fraction 1 of purified polysaccharide (purified fucoidan) derived from Nagacomb obtained in (2) of Example 1 Using the three fractions of fraction 2 and fraction 3, the growth inhibitory effect of human lymphoma monoblast-like cells (U937) was examined.
Human lymphoma monoblast-like cells (U937) are dispersed in RPM11640 medium, which is a highly nutritious liquid medium widely used for culturing mammalian cells, and cultured under an atmosphere of 5% CO _{2 at} 37 ° C. A cell suspension was obtained. In a 96-well microplate, the previously cultured cell suspension (90 μL) was dispensed to a cell density of 1.0 × 10 ⁶ / mL, and the mixture was incubated at 37 ° C. under 5% CO ₂ for 24 hours. After culturing, a solution containing each of the fractions 1 to 3 was added and cultured so that the concentrations thereof were 0, 10, 100, and 1000 μg / mL, respectively. After 48 hours or 72 hours, 10 μL of WST-PMS was added to each well and incubated at 37 ° C. for 4 hours.

Here, WST-1, which is a terazolium salt, becomes a substrate for intracellular mitochondrial dehydrogenase and is reduced by living cells to produce WST-1 formazan. The amount of WST-1 formazan released from the living cells is in good agreement with the number of living cells and produces a yellow color in the medium. By measuring the absorbance of this medium, the number of viable cells can be measured. 1-methoxy PMS works as an adjunct to WST-1 and enables measurement with higher sensitivity. Therefore, by this measurement method, the absorbance (450 nm) of each sample was measured with a microplate reader to determine the cell viability, and the cell growth inhibitory effect was evaluated.
The results of the cell viability of fractions 1 to 3 thus obtained are shown in FIG. All fractions showed significant inhibitory activity at a concentration of 100 μg / mL.

(2) Measurement of apoptosis induction effect The apoptosis induction effect was examined by the following method. First, the U937 cell suspension (4.5 mL) prepared in the above (1) was dispensed to a 60 mm dish so that the cell density was 1.0 × 10 ⁵ cells / mL, and the temperature was 37 ° C., 5% CO ₂ gas. Under the pre-culture for 24 hours, a culture solution was obtained. Thereafter, fraction 2 of the purified polysaccharide (purified fucoidan) derived from Nagacomb obtained in Example 1 was added to the culture solution (0.5 mL) to a concentration of 20 μg / mL. After culturing for 48 hours, an apoptosis cell measurement kit (APO Percentage Apotosis) using changes in the membrane structure of apoptosis cells as an index.
assay: Biocolor Ltd.) was used to measure the number of apoptotic cells. The measurement results are shown in Table 3.

Apoptotic cells increased in proportion to the concentration of fucoidan in fraction 2 added. This indicates that fucoidan induces apoptosis of U937 cells.

(3) Measurement of combined effect with other antitumor agents The antitumor activity when cysteine-β-D-arabinofuranoside, which is well known as an anticancer agent, was used in combination, was evaluated.
The U937 cell suspension (90 μL) prepared in (1) above was dispensed into a 96-well microplate so that the cell density was 1.0 × 10 ⁶ cells / mL, and the mixture was then incubated at 37 ° C., 5% CO _2. A pre-culture was performed under air for 24 hours to obtain a culture solution. Thereafter, fraction 1 of the purified polysaccharide (purified fucoidan) derived from Nagacomb obtained in Example 1 was added to this culture broth only at 1 μg / mL, and cysteine-β-D-arabinofuranoside as an antitumor agent was reduced to 0. Only 1 μM / mL, and 1 μg / mL of the same amount of Fraction 2 and 0.01 μM / mL of cysteine-β-D-arabinofuranoside were added. These solutions were incubated for 72 hours at 37 ° C. under 5% CO ₂ . After culturing, the number of U937 cells was measured using a cell number measurement kit, and the growth inhibitory activity of U937 cells was examined.
The measurement results are shown in Table 4.

  Fraction 2, which is one of the fractions of purified polysaccharide (purified fucoidan) obtained from Nagacomb, has cell growth inhibitory activity close to that of cysteine-β-D-arabinofuranoside, but this is antitumor. When used in combination with cysteine-β-D-arabinofuranoside, which is an agent, the cell viability of the culture solution is significantly smaller than when used alone, and the cell growth inhibitory effect is greater than when used alone. I understood.

  The present invention is an antitumor agent, hyaluronidase inhibitor or therapeutic agent for atopic dermatitis having excellent antitumor activity and hyaluronidase inhibitory activity. Furthermore, as an antitumor agent, antitumor activity can be synergistically enhanced by combining with other antitumor agents conventionally used.

  Therefore, the present invention is useful as an antitumor agent, a hyaluronidase inhibitor or an atopic dermatitis treatment agent, and can be made into an excellent health food by blending it into various foods and drinks. Ingestion can be expected to prevent various types of tumor suppression and atopic dermatitis.

FIG. 1 is a graph showing the hyaluronidase inhibition rate of each fraction and heparin at various dilution concentrations of sulfated polysaccharide. FIG. 2 is a graph showing IC ₅₀ of each fraction of sulfated polysaccharide and heparin. FIG. 3 is a graph showing the cell viability of each fraction at various dilution concentrations of sulfated polysaccharide.

Claims (4)

  An antitumor agent, a hyaluronidase inhibitor, or a therapeutic agent for atopic dermatitis, comprising fucoidan obtained from Nagacomb as an active ingredient.   The antitumor agent, hyaluronidase inhibitor or therapeutic agent for atopic dermatitis according to claim 1, wherein fucoidan is obtained by extracting Nagacomb with dilute hydrochloric acid aqueous solution.   Fucoidan extracts Nagacomb with dilute hydrochloric acid aqueous solution, then turns into cetylpyridinium chloride complex polysaccharide, and contains any or all of the four fractions obtained by subjecting this cetylpyridinium chloride complex polysaccharide to DEAE-Sepharose column. The antitumor agent, hyaluronidase inhibitor or therapeutic agent for atopic dermatitis according to claim 1 or 2, wherein A food or drink comprising the antitumor agent according to any one of claims 1 to 3, a hyaluronidase inhibitor, or a therapeutic agent for atopic dermatitis.

Images